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ForensicEA TutorialDid the surgeon givehepatitis C to hispatient?In a recent issue of the Journal of Medical Virology, R. Stephan Ross and colleagues (2002) report the story of a German surgeon with a viral infection. In July of 2000, the surgeon notiﬁed his hospital that he had contracted Hepatitis C Virus (HCV). HCV infects the liver, and is spread by contact with the blood of an infected person. Although many infected individuals show no symptoms, some patients suffer serious liver damage.The surgeon’s specialty was emergency orthopedic surgery. A typi-cal case might involve repairing bones and joints badly damaged in a car wreck. Orthopedic surgery requires a combination of physical Software for Evolutionary Analysis © 2002 Jon C. Herron 1 2 Did the surgeon give hepatitis C to his patient?strength and carpentry skill. It involves quick but precise work with saws, hammers, drills, pins, and screws. To a lay spectator, it can appear both violent and bloody. It is not unusual for an orthopedic sur-geon, even an unusually careful one, to cut his or her ﬁngers while working inside a patient.Among the hospital’s concerns upon learning that the surgeon had heptatis C was whether he had accidentally passed the infection to any of his patients. The hospital performed blood tests on 207 patients, three of which tested positive for HCV. Among these three, one was known to have been infected before his surgery, and another had a viral strain ...

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2Evolution by genetic driftThe ﬁrst step toward developing the tools we need to trace chains of transmission is to recognize that a viral infection is a population of indi-vidual virus particles (Figure 1). The infection may be started by one or a few particles that invade the patient’s body. But soon the invaders begin to reproduce, establishing a large population. When mutations strength and carpentry skill. It involves quick but precise work with saws, hammers, drills, pins, and screws. To a lay spectator, it can appear both violent and bloody. It is not unusual for an orthopedic sur-geon, even an unusually careful one, to cut his or her ﬁngers while working inside a patient.Among the hospital’s concerns upon learning that the surgeon had heptatis C was whether he had accidentally passed the infection to any of his patients. The hospital performed blood tests on 207 patients, three of which tested positive for HCV. Among these three, one was known to have been infected before his surgery, and another had a viral strain obviously unrelated to the surgeon’s. The last patient, however, had a strain of HCV belonging to the same subtype as the surgeon’s. This patient thus presented an open question: Did the patient get HCV from the surgeon, or did he get it from someone else?We can answer this question by reconstructing a phylogeny, or evo-lutionary tree. This tutorial, and the application ForensicEA, will help you develop the evolutionary logic needed to reconstruct trees from genetic data, and it will teach you one method for doing so. At the end we’ll give you data from the paper by Ross and colleagues. You can draw your own conclusions about the German orthopedic surgeon and his patient.?tneitap sih ot C sititapeh evig noegrus eht diD

tfird citeneg yb noitulovE occur during viral reproduction, the population becomes genetically variable. The population of virus particles can now evolve.In thinking about how a population of virus particles might evolve, we will imagine that it does so in the absence of natural selection. (This no-selection assumption is unlikely to be entirely true, but it will hold for stretches of viral genome in which the variation does not affect func-tion.) To see how populations evolve in the absence of selection, we will examine change over time in the composition of a simple model popu-lation.To see the model in action, launch the application ForensicEA. After the advertisement for Evolutionary Analysis disappears, you will see a window titled Drift (Figure 2). The box on the upper left contains our population of virus particles, living inside a patient. To get a closer look at an individual virion, click on it and hold the mouse button down. A Figure 1 A viral infection is a population of virus parti-cles. One or a few virions invade the patient to initiate tahne i nvifreioctniso nr.e pOrnocdeu icnes, iedset, ab-lishing a population. Muta-rtieopnlisc tahtiaotn oicntcruord duucrei ngge vniertailc variation. The green virion on the right is a mutant. The population of virions can now evolve.3

4 ititapeh evig noegrus eht diD ?tneitap sih ot C sFDirgifutr ew 2i ndFoowr.ensicEA’s This box shows a population of virus particles. To see a virion's nucleotide sequence, click on it and hold the mouse button down. When you click the Plot button uwinlld deir stphlea yd yatoau rb doax,t at hiins ab ox scatterplot. This box shows data you have saved. To save the nucleotide sequence for a virion, drag the virion to this box.

tfird citeneg yb noitulovEwindow will pop up showing you a picture of the virion, plus the nucle-otide sequence from a stretch of its genome that is 100 base pairs long. (The reader may notice that the nucleotide sequence is written in DNA bases. Although the hepatitis C virus is an RNA virus, we have chosen to represent its genome as a cDNA copy made for sequencing pur-poses.)Virus particles with the same color are genetically identical to each other. Note that most, if not all, of the virions in our population are black. These are genetically identical to the virion that initiated our patient’s infection. You may see a few virions of different colors. These are mutants that differ from the founder in one or more nucleotides.You can compare the sequences of two or more virions by dragging them, one at a time, to the box on the upper right. When you drag a vir-ion to this box, the box records the virion’s data. The ﬁrst column con-tains the time (in generations) at which you collected the virion. The second column shows how many differences there are between the vir-ion’s nucleotide sequence and the sequence of the ﬁrst virion in the table. The third column shows the virion’s nucleotide sequence itself.At the start of our simulation, all ﬁfty virus particles in the population are immature, or still under construction. To let the model run, click the Run button. The ﬁrst thing you will see is the immature viruses becom-ing mature. Maturation is indicated by an increase in size. Once all the virions are mature you will see them begin to reproduce.Reproduction works as follows. Every individual has an equal chance to replicate itself. We pick a mature virion at random and copy it to make the ﬁrst offspring. We then pick another adult at random and copy it to make the second offspring. We repeat this process until we have ﬁfty offspring. Some adults may be lucky and get copied more 5

6 tap sih ot C sititapeh evig noegrus eht diD ?tneithan once. Other adults may be unlucky and never get copied at all. Once we have made 50 offspring, all the adults die. Then the offspring mature and get a chance to reproduce themselves. A counter below the box keeps track of the number of generations that have passed.That’s all there is to our model. The virions have no enemies, and experience no competition. They are born, get their chance to repro-duce, then die. At ﬁrst glance, you might expect that this population will not evolve at all. There is little or no variation, and no selection. Variation and selection are necessary ingredients for adaptive evolution.The model does, however, incorporate mutation. As we have already seen, each virus particle has a genome, represented by a piece of cDNA 100 nucleotides long. Every time an adult gets copied to make an offspring, its genome gets copied too. But the copying is not perfect. Occasionally an A is subsituted for a T, or a T for a G, and so on. These mistakes, or mutations, add genetic variation to our population. When a mutation creates a new nucleotide sequence, the virion containing it gets a new color. Watch closely as the simulation runs. Most offspring are identical to their parent, but occassionally you will see new mutants appear among the offspring in the population.Our model also incorporates chance. Without selection there can be no adaptive evolution, but with chance there can be non-adaptive evo-lution. Just by luck, some genotypes may reproduce more often than others. These will become more common in the population. Also just by luck, other genotypes may reproduce less often than others. These will become rare, and may disappear altogether.If the composition of our model population changes appreciably as the generations pass, it does so because mutation creates new vari-ants, and then luck takes over. Most of the new variants disappear. But

tfird citeneg yb noitulovEoccassionally virons of a particular color (and nucleotide sequence) will have a run of good luck and become the most abundant form in the population.This mechanism of evolution is known as genetic drift. In order to examine genetic drift in more detail, we need to gather some data.Pause the simulation for a moment. If you have not already done so, drag a virion to the data box on the upper right. This will save its nucle-otide sequence. What we want to do now is run the simulation for sev-eral hundred generations, recording a nucleotide sequence every 50 to 100 generations along the way.Here is a trick to make the simulation run faster. Enter a number, say 50 or 100, in the small text box to the left of the Fast Fwd button. Now click the Fast Fwd button itself. Instead of showing you every step of the virus’s life cycle, the simulation now skips from one adult generation to the next. (Behind the scenes, the simulation is still running as before.)When the simulation stops, pick a virion at random and drag it to the data box to record its sequence. (A good way to pick a random virion is to simply take the one that landed closest to the lower right corner of the box.) Click Fast Fwd again, record the sequence for another virion, and so on. Continue letting the population evolve by genetic drift and recording data. Your goal is to gather data spanning at least 700 gener-ations, recording 10 to 20 sequences along the way. Once you have your 10 or 20 sequences spanning several hundred generations, compare each sequence to the ﬁrst one you collected. Note the number of differences between the ﬁrst sequence and each subsequent one. If you click on the Plot button, ForensicEA will display a scatterplot showing the number of sequence differences between the present viral genome and the original one (y-axis) as a function of the 7

8[ For further investigation: You may have noticed that the Reset dia-log box lets you change the population size, and it lets you change how selection acts on the new mutations that appear. You may want to do some experiments on your own to see how population size, and the pattern of selection, affect the rate at which sequence changes accu-mulate in populations. If you experiment with the effect of population size on the rate of neutral evolution, be aware that chance can play a large role in any particular run. You will have to run the simulation sev-eral times at each of several different population sizes to get a good sense of whether or not population size matters.]number of generations that have passed (x-axis). What is the pattern in your graph? Copy the graph onto your worksheet. You might also want to compare the graph to the one in Figure 19.4 in Evolutionary Analysis.Now go to the File menu and select Reset.... Click the Okay button in the window that appears. Repeat the exercise you have just com-pleted.Think about what happened in your two experiments. What was similar between them? What was different? What generalizations can you make about how populations evolve by genetic drift? If someone gave you frozen samples of virions from your patient, could you make an educated guess as to how far apart in time the samples were col-lected? Why might an evolutionary biologist think of the graphs you have prepared as molecular clocks??tneitap sih ot C sititapeh evig noegrus eht diD

tfird citeneg yb ecnegrevid noitalupoPPopulation divergence by genetic driftHaving examined genetic drift in our population of virions in some detail, we can consider what will happen when our patient infects another individual. That is, when one or a few virions move from our patient to another patient, establishing a new population. The original population and the new one will both continue to evolve by drift. Will they follow similar paths, and thus remain similar in genetic composi-tion? Or will they become steadily more distinct?Close the Drift window in ForensicEA. Go to the Simulation menu and select Divergence. This will open a new window (Figure 3).The window on the upper left shows the virus population in our already-infected patient. Use the Fast Fwd button to let the simulation run in this patient for 100 generations. This will allow the population to accumulate genetic variation representative of a well-established infec-.noitInfect the new patient by dragging one or a few virions into the box at the upper right. Determine the number of nucleotide differences between a randomly chosen virion from the ﬁrst patient and a randomly chosen virion from the second patient by dragging the virions to the small boxes at lower left. (If you have only one virion in the second patient, you can drag it back after you have noted the number of sequences differences.) Record the number of differences in the table on your worksheet.Now fast forward the simulation for 50 generations. Again sample a randomly chosen virion from each population and record the number of 9

01 ?tneitap sih ot C sititapeh evig noegrus eht diD FDiigvuerreg 3e ncFeo rweinnsdicoEw.A’s This box shows the population of virus particles in the first patient. To infect the second patient, drag one or more virions into the box at right.This box shows the population of virus particles in the second patient.To compare the nucleotide sequences of two virions, drag the virions into these boxes.

tfird citeneg yb ecnegrevid noitalupoPsequence differences. Continue fast forwarding and collecting data until you have accumulated at least 10 measures of sequence difference spanning at least 500 generations.Plot a graph on your worksheet showing the number of differences between DNA sequences versus the number of generations that have passed since the second population was established from the ﬁrst. If you had sequences of virions from two infected individuals, could you make a reasonably accurate guess about how long it has been since the virus populations in the two patients shared a common ancestor? That is, could you estimate how far in the past the two patients were connected in the chain of transmission?[For further investigation: Use the Reset... command under the File menu to start a new simulation. Experiment with the number of virions transfered to the new patient to start the new infection. Do the popula-tions diverge at an appreciably different rate if you transfer 5, or 10, or 25 virions instead of just one? Is there any effect of population size on the rate at which the populations diverge? Does the rate of divergence depend on whether mutations are neutral, beneﬁcial, or deleterious? How often do you need to transfer individuals between two popula-tions, and how many individuals do you need to transfer, to prevent the populations from diverging?]11

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